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Metolachlor is extensively used and the most persistent chloroacetamide herbicide, thereby which its metabolites have been frequently detected in soils and surface and groundwaters. Microbial degradation is predominantly responsible for the removal of metolachlor from soil and water. However, few microbial strains reported previously are highly efficient in degrading potentials for metolachlor. We isolated the fungal strain MET-F-1 from an activated sludge, characterized as Penicillium oxalicum, which could degrade 88.6% of 50 mg/L metolachlor coupled with 0.1% glucose plus 0.1% yeast extract within 384 h under optimal conditions. Compared with metabolites produced by previously isolated microorganisms, different degradation products, i.e., MOXA, M2H, and MDES, detected by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), were produced through hydrolytic and reductive dechlorination by MET-F-1. This is the first report on the degradation of metolachlor by Penicillium oxalicum sp. Furthermore, field plot experiments using the wheat bran inoculum method were performed and demonstrated good metolachlor-degrading activity of this strain. This study serves as a steppingstone to promote MET-F-1 strain usage as a promising agent for metolachlor-contaminated soil remediation.